CD8<+> cytotoxic T lymphocyte for treatment of colorectal neoplasms and preparation method thereof
A colorectal tumor, cytotoxic technology, applied in the biological field, can solve the problems of anti-tumor immune function damage, inability to completely cure tumors, etc., and achieve the effect of improving killing efficiency and preventing recurrence and metastasis.
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Embodiment 1
[0039] Example 1, CD8 for the treatment of colorectal tumors + Preparation of cytotoxic T lymphocytes
[0040] 1. CEA 610D Preparation of peptides
[0041] CEA natural epitope CEA 605-613 The sequence of is YLSGANLNL. The present invention replaces asparagine (N) with aspartic acid (D) on the 6-position, and adopts solid-phase synthesis method to synthesize new modified CEA antigen peptide (CEA 610D ), the amino acid sequence is YLSGADLNL (SEQ ID NO: 1). The antigen peptide is a polypeptide molecule with high affinity combination with MHC class I molecules.
[0042] 2. Load CEA 610D Preparation of Polypeptide B Cells
[0043] 1. Isolation and identification of B cells
[0044] B cells isolated from peripheral blood mononuclear cells (PBMC) of patients with HLA-A2 positive tumors were separated by CD20 magnetic beads to obtain high-purity (above 90%) B cells.
[0045] Through flow cytometry technology, the purity of B cells in the above sorted cells was identified by f...
Embodiment 2
[0087] Example 2, Anti-tumor in vivo experiment of CEA antigen-specific CTL cell tumor-bearing mice obtained in Example 1
[0088] BALB / C-nu / nu nude mice were provided by the Experimental Animal Laboratory of Cancer Hospital, Institute of Cancer, Chinese Academy of Medical Sciences. The age of the mice is 3-5 weeks, the body weight is 17-20 g, and they are raised in nude mice indoors under the condition of SPF.
[0089] 1. Preparation of colorectal tumor mouse model
[0090] Orthotopic transplantation method: BALB / C-nu / nu nude mice are anesthetized by intraperitoneal injection of pentobarbital sodium, take a mid-abdominal incision, identify the colorectum, and make a 2mm incision in the colorectal serosa facing the intestinal wall, as deep as For the mucosal layer, 2 grains of 1mm 3 The tumor mass (surgical specimen of human colorectal cancer with complete histology) was fixed on the mucosal layer of the intestinal wall, and then the surgical incision was sutured. The growt...
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Abstract
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